U.S. patent application number 14/126898 was filed with the patent office on 2014-08-28 for purification methods for betulonic acid and boc-lysinated betulonic acid, and organic synthesis of betulonic acid amides with piperazine derivatives.
This patent application is currently assigned to KOREA INSTITUTE OF ENERGY RESEARCH. The applicant listed for this patent is Kuck-Tack Chue, Tae-Hwan Kim, Leonid Ten. Invention is credited to Kuck-Tack Chue, Tae-Hwan Kim, Leonid Ten.
Application Number | 20140243527 14/126898 |
Document ID | / |
Family ID | 46141441 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140243527 |
Kind Code |
A1 |
Chue; Kuck-Tack ; et
al. |
August 28, 2014 |
PURIFICATION METHODS FOR BETULONIC ACID AND BOC-LYSINATED BETULONIC
ACID, AND ORGANIC SYNTHESIS OF BETULONIC ACID AMIDES WITH
PIPERAZINE DERIVATIVES
Abstract
The present invention provides a method of purifying betulonic
acid contained the reaction product of organic synthesis of a Jones
oxidation reagent and betulin extracted from the bark of a birch, a
method of preparing a piperazine betulonic acid amide derivative,
which is used as a chemical having an antibacterial function, using
the high-purity betulonic acid obtained by the purification method
and a derivative prepared by this method, a method of purifying a
Boc-lysinated betulonic acid monomer ester contained in the
reaction product of organic synthesis of lysine and the high-purity
betulonic acid (starting material) obtained by the purification
method, and a method of purifying Boc-lysinated betulonic acid
contained in the reaction product of hydrolysis of the high-purity
Boc-lysinated betulonic acid monomer ester.
Inventors: |
Chue; Kuck-Tack; (Daejeon,
KR) ; Kim; Tae-Hwan; (Daejeon, KR) ; Ten;
Leonid; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chue; Kuck-Tack
Kim; Tae-Hwan
Ten; Leonid |
Daejeon
Daejeon
Daejeon |
|
KR
KR
KR |
|
|
Assignee: |
KOREA INSTITUTE OF ENERGY
RESEARCH
Daejeon
KR
|
Family ID: |
46141441 |
Appl. No.: |
14/126898 |
Filed: |
June 29, 2012 |
PCT Filed: |
June 29, 2012 |
PCT NO: |
PCT/KR2012/005154 |
371 Date: |
December 17, 2013 |
Current U.S.
Class: |
544/294 ;
544/360; 544/380; 558/276; 562/498 |
Current CPC
Class: |
C07D 295/182 20130101;
C07J 63/008 20130101; C07D 213/74 20130101; C07D 239/42
20130101 |
Class at
Publication: |
544/294 ;
562/498; 544/380; 544/360; 558/276 |
International
Class: |
C07J 63/00 20060101
C07J063/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2011 |
KR |
10-2011-0064883 |
Claims
1. A method of purifying betulonic acid, comprising the steps of:
a) injecting a Jones oxidation reagent
(CrO.sub.3/c-H.sub.2SO.sub.4/H.sub.2O) into a mixed solution of
acetone and betulin to obtain an organic synthesis reaction product
and filtering and concentrating the obtained reaction product; b)
introducing ethyl acetate into the concentrated reaction product to
liquid-liquid extract betulonic acid c) introducing a saturated
aqueous sodium chloride solution (brine) into the ethyl acetate
solution, in which the extracted betulonic acid is dissolved, to
wash the ethyl acetate solution and remove impurities therefrom; d)
introducing hexane or petroleum ether, as a nonpolar solvent, into
the ethyl acetate solution to remove nonpolar substances, passing
the washed ethyl acetate solution through an adsorption tower
filled with aluminum oxide or silica gel, as a polar adsorbent, to
remove polar substances, and then drying the ethyl acetate solution
to produce betulonic acid (purity: more than 92%, yield to birch
bark: 2.75 wt %); and e) dissolving the produced betulonic acid in
a mixed solvent of ethanol and acetonitrile, separating and
treating the mixed solution using a semi-preparative chromatography
column filled with C.sub.18 stationary phase, and then drying the
separated and treated mixed solution to produce high-purity
betulonic acid (purity: more than 99.5%, yield to birch bark: 1.8
wt %).
2. The method of claim 1, wherein the step a) comprises the steps
of: introducing methanol into the reaction product (1) and then
filtering the reaction product to remove a blue precipitate
(Cr.sub.2O.sub.3) and obtain a dark brown filtrate (2); and
evaporating 40.about.50 vol % of the filtrate (2) to recover
methanol and acetone (3).
3. The method of claim 1, wherein the step b) comprises the steps
of: introducing ethyl acetate (5) into the concentrated reaction
product (4) at a ratio of 1:1.3.about.2 (v/v) in a liquid-liquid
extractor and then stirring the mixture to first extract betulonic
acid from an upper ethyl acetate layer (6) in the liquid-liquid
extractor; and introducing ethyl acetate (8) into a lower layer (7)
of the liquid-liquid extractor at a ratio of 1:1.about.2 (v/v) to
second extract betulonic acid from an upper ethyl acetate layer (9)
in another liquid-liquid extractor.
4. The method of claim 1, wherein the step c) comprises the steps
of: introducing a saturated aqueous sodium chloride solution
(brine) (12) into the ethyl acetate solution (11), in which
betulonic acid is dissolved, at a ratio of 2:1 (v/v) in a
liquid-liquid extractor to first separate and remove impurities
(14) from an lower layer of the liquid-liquid extractor; and
introducing a saturated aqueous sodium chloride solution (brine)
(15) into the ethyl acetate solution (13), in which the first
washed betulonic acid is dissolved, at a ratio of 3.about.5:1 (v/v)
in another liquid-liquid extractor to secondly separate and remove
water-soluble impurities (15) from an lower layer of the
liquid-liquid extractor.
5. The method of claim 1, wherein the step d) comprises the steps
of: introducing hexane or petroleum ether, as a nonpolar solvent
(18), into the washed ethyl acetate solution at a ratio of
1:1.about.5 (v/v) to precipitate green impurities, thus removing
nonpolar substances; supplying an organic solvent, in which
betulonic acid is dissolved, into at least one adsorption tower
filled with a polar adsorbent (aluminum oxide or silica gel) to
adsorb and remove impurities having higher polarity than betulonic
acid, thus removing polar substances; and evaporating and drying
the betulonic acid-containing solution having passed through the
adsorption tower to produce white crystalline betulonic acid
(purity: more than 92%, yield to birch bark: 2.75 wt %).
6. The method of claim 1, wherein the step e) is the step of:
dissolving the produced betulonic acid in a mixed solvent of
ethanol and acetonitrile (1:1 (v/v)) to obtain a sample, separating
and treating the sample while supplying an eluent of acetonitrile
and water (86:14 (v/v)) into a semi-preparative chromatography
column filled with C.sub.18 stationary phase at a flow velocity of
0.03.about.0.1 cm/sec, and then evaporating and drying the
separated and treated sample to produce white crystalline betulonic
acid (purity: more than 99.5%, yield to birch bark: 1.8 wt %).
7. A method of preparing a piperazine betulonic acid amide
derivative using high-purity betulonic acid, comprising the steps
of: dissolving betulonic acid chloride obtained from the
high-purity betulonic acid prepared by the method of claim 1 in
anhydrous dichloromethane (CH.sub.2Cl.sub.2); reacting the
dissolved betulonic acid chloride with anhydrous triethylamine and
any one selected from among 1-(4-fluorophenyl) piperazine,
1-(3,4-dichlorophenyl) piperazine, 1-(4-nitrophenyl)piperazine,
1-(2-pyridyl) piperazine, 1-(2-pyrimidyl) piperazine and
1-(4-trifluoromethylphenyl) piperazine; washing and drying the
reaction product; and fractionating the washed and dried reaction
product using a reverse-phase C.sub.18 semi-preparative
chromatography column to synthesize a piperazine betulonic acid
amide derivative.
8. The method of claim 7, wherein the synthesized piperazine
betulonic acid amide derivative is any one selected from among
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid,
4-(3',4'-dichlorophenylpiperazine-1-yl) amide betulonic acid,
4-(4'-nitrophenylpiperazine-1-yl) amide betulonic acid,
4-(2-pyridylpiperazine-1-yl) amide betulonic acid,
4-(2-pyrimidylpiperazine-1-yl) amide betulonic acid and
4-(4'-trifluoromethylphenylpiperazine-1-yl) amide betulonic
acid.
9. A piperazine betulonic acid amide derivative using high-purity
betulonic acid, the derivative being represented by Chemical
Formula 1 below: ##STR00012## wherein R is any one selected from
among 1-(4-fluorophenyl) piperazine, 1-(3,4-dichlorophenyl)
piperazine, 1-(4-nitrophenyl)piperazine, 1-(2-pyridyl) piperazine,
1-(2-pyrimidyl) piperazine and 1-(4-trifluoromethylphenyl)
piperazine.
10. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 6 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
11. The method of claim 10, wherein, in the step a), the mixed
solvent has a mixing ratio of ethylacetate:hexane (or petroleum
ether)=1:0.4.about.2.4 (v/v), and removes polar impurities
contained in the organic synthesis reaction product of a
Boc-lysinated betulonic acid monomer ester, the retention time of
the polar impurities in the reaction product being 4 minutes or
less.
12. The method of claim 10, wherein the step b) comprises the steps
of: dissolving white crystals in ethanol to make a sample having a
concentration of 10.about.16 g/L, leaving the sample at
20.about.-2.degree. C. to crystallize impurities having a retention
time of 15.+-.0.5 minutes, and then filtering the sample to first
remove the crystallized impurities; evaporating and drying a
filtrate, dissolving the filtrate in ethanol to make a sample
having a concentration of 19.about.30 g/L, leaving the sample at
20.about.-2.degree. C. to crystallize impurities having a retention
time of 15.+-.0.5 minutes, and then filtering the sample to
secondly remove the crystallized impurities; and evaporating and
drying a filtrate, dissolving the filtrate in ethanol to make a
sample having a concentration of 50.about.85 g/L, leaving the
sample at 20.about.-2.degree. C. to crystallize impurities having a
retention time of 15.+-.0.5 minutes, and then filtering the sample
to thirdly remove the crystallized impurities.
13. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 5 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
14. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 4 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
15. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 3 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
16. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 2 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
17. A method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method of claim 1 as a
starting material, comprising the steps of: a) dissolving an
organic synthesis reaction product of a Boc-lysinated betulonic
acid monomer ester in a mixed solvent of ethyl acetate and hexane
(or petroleum ether) and then treating the resultant solution with
an adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%, yield
to birch bark: 0.47 w %).
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of purifying
betuloinic acid, a method of preparing a piperazine betulonic acid
amide derivative using high-purity betulonic acid and a derivative
prepared by this method, and a method of purifying Boc-lysinated
betulonic acid using high-purity betulonic acid. More particularly,
the present invention relates to a method of purifying betulonic
acid from the organic synthesis reaction product of a Jones
oxidation reagent and betulin extracted from the bark of a birch, a
method of preparing a piperazine betulonic acid amide derivative,
which is used as a chemical having an antibacterial function, using
the high-purity betulonic acid obtained by the purification method
and a derivative prepared by this method, a method of purifying a
Boc-lysinated betulonic acid monomer ester from the organic
synthesis reaction product of lysine and the high-purity betulonic
acid (starting material) obtained by the purification method, and a
method of purifying Boc-lysinated betulonic acid from the
hydrolysis reaction product of the high-purity Boc-lysinated
betulonic acid monomer ester.
BACKGROUND ART
[0002] Betulin is used as a raw material for organic synthesis of
betulinic acid, betulonic acid or derivatives thereof.
##STR00001##
[0003] It was scientifically discovered in the paper [Pisha, E. et
al., (1995) J. M. nature Medicine, 1, 1046-1051] that betulinic
acid has antitumor activity in melanomas (for example, MEL-2, MEL-2
and MEL-4).
[0004] Further, it was published in the paper [Fujioka, T. et al.
(1994) J. Nat. Prod. 57, 243-247] that betulinic acid has anti-HIV
activity in H9 lymphocytic cells.
[0005] Further, it was published in the paper [Yunhao Gong et al.
Antiviral Research 64 (2004) 127-130] that betulin exhibits
antiviral effects upon herpes simplex viruses when it is mixed with
acyclovir.
[0006] Further, it was disclosed in PCT/US2005/039068
(WO2006/050158 A2) by GLINSKI that betulin ether derivatives were
applied to functional cosmetics in order to obtain the effects of
skin and hair protection in skin moisturizers, antiperspirants,
anti-wrinkle treatment creams, hair depilatories and the like.
[0007] Moreover, the present inventors, P. Allaway et al., have
recently insisted through U.S. Pat. No. 7,365,221B2 that
monoacylated betulin and dihydrobetulin derivatives have potent
anti-HIV activity.
[0008] Betuloinic acid is represented by the following Chemical
Formula:
##STR00002##
[0009] Betulonic acid is synthesized by Jones oxidation of betulin
extracted from the bark of a birch using an organic solvent.
Betulonic acid is not easily dissolved in a biocompatible solvent,
but has useful biological characteristics such as anti-cancer
activity, anti-inflammatory activity, etc.
[0010] Referring to related prior arts, it was disclosed in WO
2007/141398 A1 that derivatives of 28-eugenol ester betulonic acid,
28-aspartateamide dimethyl ester betulonic acid, 28-acetate
betulonic acid and the like exhibit anti-microbial effects.
[0011] Further, it was disclosed in the papers [Kobayashi et al.,
J. Org. Chem. 66:6626-6623(2001)] and [Zhao et al., J. Org. Chem.
69:270-279(2004)] and Patent document [Saxena et al.,
US2006/0154903 A1] that betulonic acid amide derivatives are
prepared by synthesis of betulonic acid and lysine. Here, these
betulonic acid amide derivatives have attracted considerable
attention since they were verified to have an effect of suppressing
the growth of prostatic cancer cells (LNCaP, PC-3, DU-145).
Boc-Lysinated betulonic acid monomer ester (referred to as
Boc-Lys-BOA monomer ester) is obtained by organic synthesis of
betulonic acid and Boc-Lys(Cbz)-OH, and Boc-Lysinated betulonic
acid (referred to as Boc-Lys-BOA), which is known to have an effect
on prostatic cancer cells in-vivo and in-vitro experiments thereof,
is hydrolysis of betulonic acid and Boc-Lys(Cbz)-OH.
[0012] In order to use the above-mentioned derivatives and
synthetic materials as medical drugs suitable for a human body,
high-purity Boc-lysinated betulonic acid monomer ester and
high-purity Boc-lysinated betulonic acid having a purity of 99% or
more must be obtained by an advanced purification method for
removing polar and nonpolar substances during a synthesis process.
However, such an advanced purification method has not been proposed
yet.
DISCLOSURE
Technical Problem
[0013] Accordingly, the present invention has been devised to solve
the above-mentioned problems, and an object of the present
invention is to provide an advanced purification method of
producing high-purity betulonic acid from the organic synthesis
reaction product of betulin and a Jones oxidation reagent.
[0014] Another object of the present invention is to provide a
method of preparing a piperazine betulonic acid amide derivative
having an antibacterial function using high-purity betulonic acid
and a derivative prepared by this method.
[0015] A further object of the present invention is to provide a
method of purifying a Boc-lysinated betulonic acid monomer ester by
removing residual reactants and by-products from the organic
synthesis reaction product of lysine and the high-purity betulonic
acid (starting material) obtained by the advanced purification
method, and a method of purifying Boc-lysinated betulonic acid from
the hydrolysis reaction product of the high-purity Boc-lysinated
betulonic acid monomer ester.
Technical Solution
[0016] In order to accomplish the above objects, an aspect of the
present invention provides a method of purifying betulonic acid,
including the steps of: a) injecting a Jones oxidation reagent
(CrO.sub.3/c-H.sub.2SO.sub.4/H.sub.2O) into a mixed solution of
acetone and betulin to obtain an organic synthesis reaction product
and filtering and concentrating the obtained reaction product; b)
introducing ethyl acetate into the concentrated reaction product to
liquid-liquid extract betulonic acid; c) introducing a saturated
aqueous sodium chloride solution (brine) into the ethyl acetate
solution, in which the extracted betulonic acid is dissolved, to
wash the ethyl acetate solution and remove impurities therefrom; d)
introducing hexane or petroleum ether, as a nonpolar solvent, into
the ethyl acetate solution to remove nonpolar substances, passing
the washed ethyl acetate solution through an adsorption tower
filled with aluminum oxide or silica gel, as a polar adsorbent, to
remove polar substances, and then drying the ethyl acetate solution
to produce betulonic acid (purity: more than 92%, yield to birch
bark: 2.75 wt %); and e) dissolving the produced betulonic acid in
a mixed solvent of ethanol and acetonitrile, separating and
treating the mixed solution using a semi-preparative chromatography
column filled with C.sub.18 stationary phase, and then drying the
separated and treated mixed solution to produce high-purity
betulonic acid (purity: more than 99.5%, yield to birch bark: 1.8
wt %).
[0017] The step a) may include the steps of: introducing methanol
into the reaction product (1) and then filtering the reaction
product to remove a blue precipitate (Cr.sub.2O.sub.3) and obtain a
dark brown filtrate (2); and evaporating 40.about.50 vol % of the
filtrate (2) to recover methanol and acetone (3).
[0018] The step b) may include the steps of: introducing ethyl
acetate (5) into the concentrated reaction product (4) at a ratio
of 1:1.3.about.2 (v/v) in a liquid-liquid extractor and then
stirring the mixture to first extract betulonic acid from an upper
ethyl acetate layer (6) in the liquid-liquid extractor; and
introducing ethyl acetate (8) into a lower layer (7) of the
liquid-liquid extractor at a ratio of 1:1.about.2 (v/v) to second
extract betulonic acid from an upper ethyl acetate layer (9) in
another liquid-liquid extractor.
[0019] The step c) may include the steps of: introducing a
saturated aqueous sodium chloride solution (brine) (12) into the
ethyl acetate solution (11), in which betulonic acid is dissolved,
at a ratio of 2:1 (v/v) in a liquid-liquid extractor to first
separate and remove impurities (14) from an lower layer of the
liquid-liquid extractor; and introducing a saturated aqueous sodium
chloride solution (brine) (15) into the ethyl acetate solution
(13), in which the first washed betulonic acid is dissolved, at a
ratio of 3.about.5:1 (v/v) in another liquid-liquid extractor to
secondly separate and remove water-soluble impurities (15) from an
lower layer of the liquid-liquid extractor.
[0020] The step d) may include the steps of: introducing hexane or
petroleum ether, as a nonpolar solvent (18), into the washed ethyl
acetate solution at a ratio of 1:1.about.5 (v/v) to precipitate
green impurities, thus removing nonpolar substances; supplying an
organic solvent, in which betulonic acid is dissolved, into an
adsorption tower filled with at least one polar adsorbent (aluminum
oxide or silica gel) to adsorb and remove impurities having higher
polarity than betulonic acid, thus removing polar substances; and
evaporating and drying the betulonic acid-containing solution
having passed through the adsorption tower to produce white
crystalline betulonic acid (purity: more than 92%, yield to birch
bark: 2.75 wt %).
[0021] The step e) may be the step of: dissolving the produced
betulonic acid in a mixed solvent of ethanol and acetonitrile (1:1
(v/v)) to obtain a sample, separating and treating the sample while
supplying an eluent of acetonitrile and water (86:14 (v/v)) into a
semi-preparative chromatography column filled with C.sub.18
stationary phase at a flow velocity of 0.03.about.0.1 cm/sec, and
then evaporating and drying the separated and treated sample to
produce white crystalline betulonic acid (purity: more than 99.5%,
yield to birch bark: 1.8 wt %).
[0022] Another aspect of the present invention provides a method of
preparing a piperazine betulonic acid amide derivative using
high-purity betulonic acid, including the steps of: dissolving
betulonic acid chloride obtained from the high-purity betulonic
acid prepared by the method of any one of claims 1 to 6 in
anhydrous dichloromethane (CH.sub.2Cl.sub.2); reacting the
dissolved betulonic acid chloride with anhydrous triethylamine and
any one selected from among 1-(4-fluorophenyl) piperazine,
1-(3,4-dichlorophenyl) piperazine, 1-(4-nitrophenyl)piperazine,
1-(2-pyridyl) piperazine, 1-(2-pyrimidyl) piperazine and
1-(4-trifluoromethylphenyl) piperazine; washing and drying the
reaction product; and fractionating the washed and dried reaction
product using a reverse-phase C.sub.18 semi-preparative
chromatography column to synthesize a piperazine betulonic acid
amide derivative.
[0023] The piperazine betulonic acid amide derivative synthesized
in the step b) may be any one selected from among
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid,
4-(3',4'-dichlorophenylpiperazine-1-yl) amide betulonic acid,
4-(4'-nitrophenylpiperazine-1-yl) amide betulonic acid,
4-(2-pyridylpiperazine-1-yl) amide betulonic acid,
4-(2-pyrimidylpiperazine-1-yl) amide betulonic acid and
4-(4'-trifluoromethylphenylpiperazine-1-yl) amide betulonic
acid.
[0024] Still another aspect of the present invention provides a
piperazine betulonic acid amide derivative using high-purity
betulonic acid, the derivative being represented by Chemical
Formula 1 below:
##STR00003##
[0025] wherein R is any one selected from among 1-(4-fluorophenyl)
piperazine, 1-(3,4-dichlorophenyl) piperazine,
1-(4-nitrophenyl)piperazine, 1-(2-pyridyl) piperazine,
1-(2-pyrimidyl) piperazine and 1-(4-trifluoromethylphenyl)
piperazine.
[0026] Still another aspect of the present invention provides a
method of purifying Boc-lysinated betulonic acid using the
high-purity betulonic acid obtained by the method as a starting
material, including the steps of: a) dissolving an organic
synthesis reaction product of a Boc-lysinated betulonic acid
monomer ester in a mixed solvent of ethyl acetate and hexane (or
petroleum ether) and then treating the resultant solution with an
adsorption tower filed with aluminum oxide (or silica gel) to
remove polar impurities; b) evaporating and drying the solution
discharged from the adsorption tower to obtain white crystals,
dissolving the white crystals in ethanol, crystallizing impurities
in the resultant solution at 20.about.-2.degree. C., and then
filtering this solution to remove the crystallized impurities; c)
evaporating and drying the filtrate to obtain crystals, dissolving
the crystals in ethanol, and then fractionating the resultant
solution using a reverse phase C.sub.18 chromatography to obtain a
Boc-lysinated betulonic acid monomer ester having a purity of 99%
or more (yield to birch bark: 0.58 w %); and (d) dissolving the
Boc-lysinated betulonic acid monomer ester in dichloromethane (DCM)
or tetrahydrofuran (THF), adding sodium hydroxide (NaOH), lithium
hydroxide (LiOH) or potassium hydroxide (KOH) thereto, hydrolyzing
the resultant solution, and then fractionating the hydrolysis
reaction product using a reverse phase C.sub.18 chromatography to
obtain Boc-lysinated betulonic acid (purity: more than 99.5%. yield
to birch bark: 0.47 w %).
[0027] In the step a), the mixed solvent may have a mixing ratio of
ethylacetate:hexane (or petroleum ether)=1:0.4.about.2.4 (v/v), and
may remove polar impurities contained in the organic synthesis
reaction product of a Boc-lysinated betulonic acid monomer ester,
the retention time of the polar impurities in the reaction product
being 4 minutes or less.
[0028] The step b) may include the steps of: dissolving white
crystals in ethanol to make a sample having a concentration of
10.about.16 g/L, leaving the sample at 20.about.-2.degree. C. to
crystallize impurities having a retention time of 15.+-.0.5
minutes, and then filtering the sample to first remove the
crystallized impurities; evaporating and drying a filtrate,
dissolving the filtrate in ethanol to make a sample having a
concentration of 19.about.30 g/L, leaving the sample at
20.about.-2.degree. C. to crystallize impurities having a retention
time of 15.+-.0.5 minutes, and then filtering the sample to
secondly remove the crystallized impurities; and evaporating and
drying a filtrate, dissolving the filtrate in ethanol to make a
sample having a concentration of 50.about.85 g/L, leaving the
sample at 20.about.-2.degree. C. to crystallize impurities having a
retention time of 15.+-.0.5 minutes, and then filtering the sample
to thirdly remove the crystallized impurities.
Advantageous Effects
[0029] As described above, the present invention provides an
advanced purification technology of producing high-purity betulonic
acid (purity: 99.5%, yield to birch bark: 1.8 wt %) from the
organic synthesis reaction product of betulin and a Jones oxidation
reagent.
[0030] Further, the present invention provides a technology of
preparing a piperazine betulonic acid amide derivative, which is a
betulonic acid derivative having an antibacterial function as a
chemical drug, using the high-purity betulonic acid produced by the
advanced purification technology.
[0031] Further, the present invention provides a technology of
purifying a Boc-lysinated betulonic acid monomer ester by removing
residual reactants and side-products from the organic synthesis
reaction product of lysine and the high-purity betulonic acid
(starting material) obtained by the advanced purification
technology, and a technology of purifying Boc-lysinated betulonic
acid from the hydrolysis reaction product of the high-purity
Boc-lysinated betulonic acid monomer ester to produce high-purity
betulonic acid (purity: more than 99.5%, yield to birch bark: 0.47
wt %).
[0032] Consequently, these technologies can be practically used in
methods of producing therapeutic agents for an androgen-dependent
prostatic cancer cells (LNCaP) and androgen-independent prostatic
cancer cells (PC-3 and DU-145), thus conferring great expectations
for industrial applicability.
DESCRIPTION OF DRAWINGS
[0033] FIG. 1a is a schematic view showing a purification method of
producing high-purity betulonic acid according to an embodiment of
the present invention, and FIG. 1b is a view showing piperazine
betulonic acid derivatives using the high-purity betulonic
acid;
[0034] FIG. 2a is a schematic view showing a process of purifying a
Boc-lysinated betulonic acid monomer ester from the organic
synthesis reaction product of lysine and the high-purity betulonic
acid according to an embodiment of the present invention, and FIG.
2b is a schematic view showing a process of purifying Boc-lysinated
betulonic acid from the hydrolysis reaction product of the
high-purity Boc-lysinated betulonic acid monomer ester according to
an embodiment of the present invention;
[0035] FIG. 3 is graph of HPLC analysis of the organic synthesis
reaction product of a Boc-lysinated betulonic acid monomer ester
(Boc-Lys-BOA monomer ester) according to an embodiment of the
present invention;
[0036] FIG. 4 is graph of HPLC analysis of a Boc-lysinated
betulonic acid monomer ester (Boc-Lys-BOA monomer ester) according
to an embodiment of the present invention; and
[0037] FIG. 5 is graph of HPLC analysis of Boc-lysinated betulonic
acid (Boc-Lys-BOA) according to an embodiment of the present
invention.
BEST MODE
[0038] The constitutions, functions and advantages of the present
invention will be more clearly understood from the following
detailed description of preferred embodiments taken in conjunction
with the accompanying drawings. Further, in the description of the
present invention, when it is determined that the detailed
description of the related art would obscure the gist of the
present invention, the description thereof will be omitted.
[0039] FIG. 1a is a schematic view showing a purification method of
producing high-purity betulonic acid according to an embodiment of
the present invention, and FIG. 1b is a view showing piperazine
betulonic acid derivatives using the high-purity betulonic
acid.
[0040] In the purification method of producing high-purity
betulonic acid according to the present invention, basically,
betulin is dissolved in acetone and then chemically with a Jones
oxidation reagent (CrO.sub.3/c-H.sub.2SO.sub.4/H.sub.2O) to convert
betulin into betulonic acid, methanol is introduced into the
resultant solution to precipitate impurities included in the
reaction product, and then betulonic acid is extracted into an
organic solvent layer. This organic solvent layer contains
Cr.sub.2O.sub.3, water, acetone, methanol, unreacted betulin and
by-products as well as betulonic acid. Therefore, in order for
betulonic acid to be used as a raw material for synthesizing
derivatives having biological activity, an advanced purification
method of effectively removing unreacted betulin and impurities
according to the present invention is required.
[0041] Hereinafter, a purification method of producing high-purity
betulonic acid according to the present invention will be
stepwisely described in detail with reference to FIG. 1.
[0042] First, a Jones oxidation reagent
(CrO.sub.3/c-H.sub.2SO.sub.4/H.sub.2O) is injected into a mixed
solution of acetone and betulin to obtain an organic synthesis
reaction product (1).
[0043] Subsequently, methanol is introduced into the reaction
product (1), and then the reaction product (1) is filtered to
remove blue impurities and obtain a dark brown filtrate (2).
[0044] Subsequently, 40.about.50 vol % of the filtrate (2) is
evaporated to recover methanol and acetone (3) and obtain a
concentrate (4).
[0045] Subsequently, ethyl acetate (5) is added to the concentrate
(4) in a liquid-liquid extractor at a ratio of concentrate
(4):ethyl acetate (5)=1:1.3.about.2 (v/v), at which layer
separation easily occurs, and then the mixture is sufficiently
stirred to first extract betulonic acid from an upper ethyl acetate
layer (6) of the liquid-liquid extractor.
[0046] Subsequently, in order to recover betulonic acid existing in
a lower layer (7) of the liquid-liquid extractor, ethyl acetate (8)
is mixed with the lower layer (7) in another liquid-liquid
extractor at a ratio of lower layer (7):ethyl acetate
(8)=1:1.about.2 (v/v), at which layer separation easily occurs, and
then the mixture is sufficiently stirred to secondly extract
betulonic acid from an upper ethyl acetate layer (9) of the other
liquid-liquid extractor.
[0047] A mixed ethyl acetate layer (11) of the ethyl acetate layer
(6) containing the first extracted betulonic acid and the ethyl
acetate layer (9) containing the second extracted betulonic acid is
put into another liquid-liquid extractor at ratio of ethyl acetate
(11):saturated aqueous sodium hydroxide solution (brine) (12)=2:1
(v/v), at which catalyst suspended solids such as Cr.sub.2O.sub.3
and the like are most easily removed in an organic solvent layer,
and then first washed with the saturated aqueous sodium hydroxide
solution (brine) (12) to first separate and remove impurities (14)
from the lower layer of the liquid-liquid extractor.
[0048] Subsequently, the first washed ethyl acetate layer (13), in
which betulonic acid is dissolved, is put into another
liquid-liquid extractor at ratio of ethyl acetate (13):saturated
aqueous sodium hydroxide solution (brine) (15)=2:1 (v/v) at which
catalyst suspended solids such as Cr.sub.2O.sub.3 and the like are
most easily removed in an organic solvent layer, and then second
washed with the saturated aqueous sodium hydroxide solution (brine)
(15) to second remove water-soluble impurities (17) and separate an
upper ethyl acetate layer (16) from the lower layer of the
liquid-liquid extractor.
[0049] Subsequently, hexane or petroleum ether, as a nonpolar
solvent (18), is introduced into the second washed ethyl acetate
layer (16) at a ratio of ethyl acetate:hexane or petroleum
ether=1:1.about.5 (v/v), at which the selectivity of impurities and
betulonic acid in a polar adsorbent is high, to precipitate green
impurities, thus removing nonpolar substances.
[0050] Subsequently, an organic solvent (19, 20), in which
betulonic acid is dissolved, is supplied into at least one
adsorption tower filled with a polar adsorbent (aluminum oxide or
silica gel) to adsorb and remove impurities having higher polarity
than betulonic acid, thus removing polar substances.
[0051] Subsequently, a first solution, which was discharged from
one adsorption tower (30), is separately collected in an amount of
5.about.10 fold of the volume of the adsorption tower 30, and then
a second solution, which was washed with a mixed solvent (21) of
pure ethyl acetate and hexane (or petroleum ether) to recover
betulonic acid existing in the pores of another adsorption tower
(30), is mixed with the first solution, and then the mixed solution
is completely evaporated and dried to produce white crystalline
betulonic acid (purity: more than 92%, yield to birch bark: 2.75 wt
%).
[0052] Subsequently, the white crystalline betulonic acid is
dissolved in a mixed solvent of ethanol and acetonitrile
(ethanol:acetonitrile=1:1 (v/v), at a ratio of which solubility of
the white crystalline betulonic acid is maximized) while it being
not split and pecked, thus making a betulonic acid solution (28)
having a concentration of 25.about.50 mg/L, and then the betulonic
acid solution (28) is injected into a semi-preparative
chromatography column filled with C.sub.18 stationary phase at a
flow velocity of 0.03.about.0.1 cm/sec while supplying acetonitrile
and water as an eluent into the semi-preparative chromatography
column at a flow velocity of 0.03.about.0.1 cm/sec.
[0053] Finally, the betulonic acid solution (29) discharged from
the semi-preparative chromatography column is completely evaporated
and dried to produce white crystalline betulonic acid (purity: more
than 99.5%, yield to birch bark: 1.8 wt %).
[0054] Through the above procedures, high-purity betulonic acid is
produced.
[0055] Further, the present invention provides a method of
preparing a betulonic acid derivative having an antibacterial
activity using the high-purity betulonic acid. This method includes
the following steps. In 1.sup.st step, betulonic acid reacts with
oxalyl chloride to obtain betulonic acid chloride 1 of FIG. 1 as an
intermediate material.
##STR00004##
[0056] In the 2.sup.nd step, betulonic acid chloride 1 is dissolved
in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then reacts
with anhydrous triethylamine and 1-(4-fluorophenyl) piperazine at
15.about.25.degree. C. at which precipitation does not occur. Then,
the reaction product is washed with HCl and H.sub.2O, dried with
Na.sub.2SO.sub.4 and then evaporated to obtain crystals. Then, the
crystals are fractionated using a reverse-phase C.sub.18
semi-preparative chromatography column to obtain
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid 2 of FIG.
1.
[0057] Or, in the 2.sup.nd step, betulonic acid chloride 1 is
dissolved in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then
reacts with anhydrous triethylamine and 1-(3,4-dichlorophenyl)
piperazine at 15.about.25.degree. C. at which precipitation does
not occur. Then, the reaction product is washed with HCl and
H.sub.2O, dried with Na.sub.2SO.sub.4 and then evaporated to obtain
crystals. Then, the crystals are fractionated using a reverse-phase
C.sub.18 semi-preparative chromatography column to obtain
4-(3',4'-dichlorophenylpiperazine-1-yl) amide betulonic acid 3 of
FIG. 1.
[0058] Or, in the 2.sup.nd step, betulonic acid chloride 1 is
dissolved in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then
reacts with anhydrous triethylamine and 1-(4-nitrophenyl)piperazine
at 15.about.25.degree. C. at which precipitation does not occur.
Then, the reaction product is washed with HCl and H.sub.2O, dried
with Na.sub.2SO.sub.4 and then evaporated to obtain crystals. Then,
the crystals are fractionated using a reverse-phase C.sub.18
semi-preparative chromatography column to obtain
4-(4'-nitrophenylpiperazine-1-yl) amide betulonic acid 4 of FIG.
1.
[0059] Or, in the 2.sup.nd step, betulonic acid chloride 1 is
dissolved in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then
reacts with anhydrous triethylamine and 1-(2-pyridyl) piperazine at
15.about.25.degree. C. at which precipitation does not occur. Then,
the reaction product is washed with HCl and H.sub.2O, dried with
Na.sub.2SO.sub.4 and then evaporated to obtain crystals. Then, the
crystals are fractionated using a reverse-phase C.sub.18
semi-preparative chromatography column to obtain 4-(2-pyridyl
piperazine-1-yl) amide betulonic acid 5 of FIG. 1.
[0060] Or, in the 2.sup.nd step, betulonic acid chloride 1 is
dissolved in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then
reacts with anhydrous triethylamine and 1-(2-pyrimidyl)piperazine
at 15.about.25.degree. C. at which precipitation does not occur.
Then, the reaction product is washed with HCl and H.sub.2O, dried
with Na.sub.2SO.sub.4 and then evaporated to obtain crystals. Then,
the crystals are fractionated using a reverse-phase C.sub.18
semi-preparative chromatography column to obtain 4-(2-pyrimidyl
piperazine-1-yl) amide betulonic acid 6 of FIG. 1.
[0061] Or, in the 2.sup.nd step, betulonic acid chloride 1 is
dissolved in anhydrous dichloromethane (CH.sub.2Cl.sub.2) and then
reacts with anhydrous triethylamine and 1-(4-trifluoromethylphenyl)
piperazine at 15.about.25.degree. C. at which precipitation does
not occur. Then, the reaction product is washed with HCl and
H.sub.2O, dried with Na.sub.2SO.sub.4 and then evaporated to obtain
crystals. Then, the crystals are fractionated using a reverse-phase
C.sub.18 semi-preparative chromatography column to obtain
4-(4'-trifluoromethylphenylpiperazine-1-yl) amide betulonic acid 7
of FIG. 1.
[0062] Further, the present invention provides a purification
method of preparing high-purity Boc-lysinated betulonic acid
monomer ester by removing residual reactants and by-products from
the organic synthesis reaction product of the obtained high-purity
betulonic acid, and a method of producing high-purity Boc-lysinated
betulonic acid from the hydrolysis reaction product of the prepared
high-purity Boc-lysinated betulonic acid monomer ester.
Hereinafter, these methods will be described in detail.
[0063] The organic synthesis of Boc-lysinated betuloni acid is
disclosed in the documents Kobayashi et al., J. Org. Chem.
66:6626-6623(2001), Zhao et al., J. Org. Chem. 69:270-279(2004),
and Saxena et al., US2006/0154903 A1. The organic synthesis of
Boc-lysinated betuloni acid includes the following steps.
[0064] In the 1.sup.st step, the high-purity betulonic acid
obtained the method of the present invention reacts with
lysine(Boc-Lys(Cbz)-OH) whose primary amine group and secondary
amine group are protected by benzyloxycarbonyl(Cbz) and
butyloxycarbonyl(Boc) to obtain Boc-Lys(Cbz)-OMe, which is a methyl
ester.
[0065] In the 2.sup.nd step, the Cbz(bezoyloxycarbonyl) protecting
the primary amine group of the obtained lysine(Boc-Lys(Cbz)-OH) is
detached from the Boc-Lys(Cbz)-OMe to obtain Boc-Lys-OMe.
[0066] In the 3.sup.rd step, the obtained Boc-Lys-OMe is bonded to
a carboxyl group (--COOH) of betulonic acid C-28 to synthesize
Boc-lysinated-betulonic acid monomer ester (Boc-Lys-BOMe).
[0067] In this case, Boc-Lys-BOMe is synthesized by activating the
carboxyl group (--COOH) of betulonic acid C-28 in CH.sub.2Cl.sub.2
or tetrahydrofuran using 1-hydroxybezotriazole hydrate(HOBt) and
1,3-dicyclohexyl carbodiimide (DCC) catalysts and then bonding the
activated carboxyl group (--COOH) thereof to Boc-Lys-OMe. Here, the
synthesized Boc-Lys-BOMe contains various impurities such as
Boc-Lys-BOMe, HOBt, DCC, unreacted Boc-Lys(Cbz)-OH, etc. FIG. 3
shows the results of analysis of such an organic synthesis reaction
product using HPLC (Nova-Pak C.sub.18 4 .mu.m, 3.9.times.300 mm,
UV/VIS @ 212 nm) under the gradient elution conditions given in
Table 1 below.
TABLE-US-00001 TABLE 1 Conditions of HPLC analysis of organic
synthesis reaction product of Boc-lysinated betulonic acid monomer
ester HPLC column: Nova-Pak C.sub.18 4 .mu.m, 3.9 .times. 300 mm
Detector: UV/VIS @ 212 nm Time Flow velocity Eluent A (MeCM, Eluent
B (H.sub.2O, (min) (mL/min) 0.05% TFA) 0.05% TFA) 0~4 0.6 86 14 4~6
0.6 86 .fwdarw. 98 14 .fwdarw. 2 6~26 0.6 98 2 26~28 0.6 98
.fwdarw. 86 2 .fwdarw. 14 28~ 0.6 86 14
[0068] From FIG. 3, it can be seen that the organic synthesis
reaction product includes, based on an area ratio: 50.39% and 3.1%
of unconfirmed components having their respective retention times
of 3.5 min and 4.1 min; 7.85% of Boc-lysinated betulonic acid
monomer ester (Boc-Lys-OMe); and 32.68%, 2.97% and 2.31% of
components having their respective retention times of 15.15 min,
16.08 min and 23.23 min. The organic synthesis reaction product is
purified to obtain high-purity Boc-Lys-OMe, and this high-purity
Boc-Lys-OMe can be used to produce Boc-yysinated betulonic acid.
For this purpose, advanced technologies of purifying the organic
synthesis reaction product are required.
[0069] Hereinafter, the purification method according to the
present invention will be described in more detail with reference
to FIG. 2.
[0070] FIG. 2a is a schematic view showing a process of purifying a
Boc-lysinated betulonic acid monomer ester contained in the
reaction product of organic synthesis of lysine and the high-purity
betulonic acid according to an embodiment of the present invention,
and FIG. 2b is a schematic view showing a process of purifying
Boc-lysinated betulonic acid contained in the reaction product of
hydrolysis of the high-purity Boc-lysinated betulonic acid monomer
ester according to an embodiment of the present invention.
[0071] As shown in FIG. 2, the solvent of the organic synthesis
reaction product (51) of Boc-lysinated betulonic acid monomer ester
is partially evaporated and then filtered to remove white
impurities having a retention time of 3.5.about.4.1 min of FIG. 3.
Then, tetrahydrofuran and the like included in the filtrate (53)
are evaporated and thus removed to obtain yellow solids (54). Then,
these yellow solids (54) are dissolved in a mixed solvent of ethyl
acetate and hexane (or petroleum ether) (ratio of ethyl
acetate:hexane (or petroleum ether)=1:0.4.about.2.4 (v/v), at which
the selectivity of yellow solids to a polar adsorbent is high at
the time of adsorbing and removing yellow solids using the polar
adsorbent) to obtain a sample. Then, the sample is supplied into an
adsorption tower (90) filled with aluminum oxide (or silica gel) to
adsorb and remove polar impurities having a retention time of
3.5.about.4.1 min.
[0072] A solution (58) directly discharged from the adsorption
tower (90) is mixed with a solution (59) discharged from another
adsorption tower (90) after being washed with the mixed solvent of
ethyl acetate and hexane (or petroleum ether) (ratio of ethyl
acetate:hexane (or petroleum ether)=1:0.4.about.2.4 (v/v)), and
then this mixed solution is evaporated and dried to obtain white
crystals 60.
[0073] Subsequently, the white crystals (60) are dissolved in
ethanol (EtOH) to make a sample having a concentration of
10.about.16 g/L which experimentally approximates saturated
solubility, and then the sample is left at 20.about.-2.degree. C.
to crystallize impurities having a retention time of 15 min (error
range.+-.0.5 minutes, which is changed within this range depending
on the degree of contamination of a column, the lifespan of a
column, the purity of an eluent or the like even if analysis
conditions are accurate), and then filtered to first remove the
crystallized impurities (62). Subsequently, the filtrate (63) is
evaporated and dried again and then dissolved in ethanol (EtOH) to
make another sample (64) having a concentration of 19.about.30 g/L,
and then the sample is left at 20.about.-2.degree. C. to
crystallize impurities having a retention time of 15.+-.0.5
minutes, and then the sample is filtered to secondly remove the
crystallized impurities (65). Finally, the filtrate (66) is
evaporated and dried and then dissolved in ethanol (EtOH) to make
another sample having a concentration of 50.about.85 g/L, and then
the sample is left at 20.about.-2.degree. C. to crystallize
impurities having a retention time of 15.+-.0.5 minutes, and then
the sample is filtered to thirdly remove the crystallized
impurities (68).
[0074] The filtrate (69) is completely evaporated and dried and
then dissolved in ethanol (EtOH) or a mixed solvent of acetonitrile
and water (H.sub.2O) to obtain another sample (72), and then this
sample (72) is injected into a reverse phase C.sub.18
chromatography column. Then, a solution containing Boc-lysinated
betulonic acid monomer ester, discharged from the reverse phase
C.sub.18 chromatography column, is separately collected and then
evaporated to obtain white crystals (74) having a purity of 99% or
more (purity of Boc-lysinated betulonic acid monomer ester: more
than 99%, yield to birch bark: 0.58 w %).
[0075] Finally, the white crystals (74) are dissolved in
dichloromethane (DCM) or tetrahydrofuran (THF), and then the
resultant solution is hydrolyzed by the addition of sodium
hydroxide (NaOH), lithium hydroxide (LiOH) or potassium hydroxide
(KOH) to detach a methyl group, and then the hydrolysis reaction
product is fractionated using a reverse phase C.sub.18
chromatography to obtain Boc-lysinated betulonic acid (purity: more
than 99.5%, yield to birch bark: 0.47 w %).
MODE FOR INVENTION
[0076] Hereinafter, the present invention will be described in more
detail with reference to the following Examples.
Example 1
[0077] 45 g of betulin and 1100 mL of acetone were put into a
reactor, and then uniformly stirred at 10.degree. C. A Jones
oxidation reagent (CrO.sub.3 97 g, 310 mL, c-H.sub.2SO.sub.4 65 mL)
was put into a funnel, and then introduced into the reactor filled
with betulin and acetone through the funnel over 60 min.
[0078] Subsequently, the temperature of the reactor was maintained
at 25.degree. C., and the mixed solution was stirred for 1 hour to
convert betulin into betulonic acid, and then 1000 mL of methanol
was put into the reactor. Thus, an organic synthesis reaction was
conducted. The subsequent procedures were conducted as follows
according to the purification method of FIG. 1.
[0079] The organic synthesis reaction product (1) was passed
through a glass frit filter to remove blue impurities
(Cr.sub.2O.sub.3) and obtain 2000 mL of a filtrate (pH 3) (2).
[0080] Acetone and methanol (3) were evaporated by a vacuum
evaporator to concentrate the filtrate (2) in an amount of 1300
mL.
[0081] 1300 mL of the concentrated filtrate (4) was mixed with 1800
mL of ethyl acetate (5), and then the mixture was sufficiently
stirred and then left until layer separation occurred.
[0082] The resulting mixture was separated into 2500 mL of an upper
ethyl acetate (EtOAc) layer (6) and 600 mL of a lower layer (7)
containing methanol, acetone and water.
[0083] 600 mL of the lower layer (7) was mixed with 500 mL of ethyl
acetate to form another upper ethyl acetate layer (9), and then
betulonic acid was separated from the upper ethyl acetate layer (9)
and mixed with the first ethyl acetate extract to obtain 3350 mL of
an ethyl acetate solution. The ethyl acetate solution was first
washed with 1750 mL of a saturated aqueous sodium hydroxide
solution (brine) (12) to extract residual impurities (14) and polar
substances.
[0084] 2850 mL of the upper ethyl acetate layer (13) was secondly
washed with 850 mL of a saturated aqueous sodium hydroxide solution
(brine) (15), and 3200 mL of the upper ethyl acetate solution
containing betulonic acid as a major component is treated by an
adsorption tower (30) filled with aluminum oxide.
[0085] 500 ml of the ethyl acetate extract was mixed with 1500 ml
of hexane (18) at a ratio of EtOAc:hexane=1:3 to obtain a matrix
solution having low polarity, and the matrix solution was supplied
into an adsorption tower (30) filled with 170 g of aluminum oxide
at a flow velocity of 5 mL/min, and simultaneously 7.45 BV (1
BV=volume of adsorption tower) of an initial solution (23)
discharged from the adsorption tower was discarded, and 7.5 BV of a
subsequent solution discharged therefrom was collected and then
completely evaporated and dried to 2.715 g of white crystals
(betulonic acid: 91.99% HPLC, yield to birch bark: 2.45 wt %).
[0086] Thereafter, 1.2 BV of a mixed solvent (21) of
EtOAc:hexane=1:3 (v/v) is supplied to a semi-preparative
chromatography column at a flow velocity of 5 mL/min to recover
betulonic acid existing in the pores of the adsorption tower.
[0087] The solution (25) discharged from the semi-preparative
chromatography column during the washing procedure was completely
evaporated and dried to obtain 0.308 g of white crystals (betulonic
acid: 87.43%, yield to birch bark: 0.278 wt %).
[0088] 5 to 9 ml of a matrix solution (25 g/L) prepared by
dissolving 1 g of white betulonic acid crystals in 20 mL of ethanol
and 20 mL of acetonitrile was introduced into a RP C.sub.18
semi-preparative chromatography column. Then, a betulonic acid
fraction was collected from the column while observing signals
detected by UV of 212 nm, and then evaporated and dried to obtain
0.6674 g of white betulonic acid crystals (29) having a purity of
99.69.about.99.10% (yield to birch bark: 1.8 wt %).
[0089] Or, a matrix solution (50 g/L) prepared by dissolving 2 g of
the white betulonic acid crystals purified with the column filled
with aluminum oxide in 20 mL of ethanol and 20 mL of acetonitrile
was purified by RP C.sub.18 semi-preparative chromatography column
to obtain 1.0864 g of white betulonic acid crystals (29) having a
purity of 96.54.about.97.29 (yield to birch bark: 1.43 wt %).
##STR00005##
[0090] .sup.1H NMR (600 MHz, C.sub.5D.sub.5N):
[0091] .delta.=4.74 (1H, s, H-29a), 4.62 (1H, s, H-29b), 3.03 (2H,
m, H-2), 1.70 (3H, s, H-30), 1.44 (3H, s, H-27), 1.07 (3H, s,
H-26), 1.04 (3H, s, H-23), 1.05 (3H, s, H-27), 0.99, 0.98 (3H, s,
H-25), 0.93 (3H, s, H-25)
[0092] .sup.13C NMR (125 MHz, C.sub.5D.sub.5H):
[0093] .delta.=218.55 (C3), 182.80 (C28), 150.52 (C20), 109.98
(C29), 56.62 (C17), 55.62 (C5), 50.03 (C9), 49.36 (C18), 47.54
(C4), 47.10 (C19), 42.68 (C14), 40.82 (C8), 39.80 (C1), 38.71
(C13), 37.25 (C10), 37.11 (C22), 34.33 (C2), 33.78 (C7), 32.31
(C16), 30.75 (C15), 29.88 (C21), 26.84 (C23), 25.67 (C12), 21.56
(C11), 21.20 (C24), 19.82 (C6), 19.57 (C30), 16.17 (C25), 16.02
(C26), 14.83 (C27).
Example 2
[0094] 0.55 g (1.16 mmol) of betulonic acid chloride was dissolved
in 35 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.65 mL (4.64 mmol) of anhydrous triethylamine and 0.42 g (2.32
mmol) of 1-(4-fluorophenyl) piperazine were added thereto, and then
stirred at 25.degree. C. for 48 hours.
[0095] The resulting product was washed with 10% HCl (40
mL.times.3), further washed with pure water (40 mL.times.3), and
then dried by 10 g of anhydrous Na.sub.2SO.sub.4 to remove a
solvent therefrom.
[0096] Dichloromethane (DCM) was evaporated from the washed and
dried product to obtain 0.72 g of light brown solids, these light
brown solids were dissolved in ethanol to prepare a sample, and
then this sample was introduced into RP C.sub.18 semi-preparative
chromatography column to separate a
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid fraction.
Then, this fraction was evaporated and dried to obtain 0.63 g of
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid (yield:
89.8%).
[0097] C.sub.40H.sub.57FN.sub.2O.sub.2
4-(4'-fluorophenylpiperazine-1-yl) amide betulonic acid 2
##STR00006##
[0098] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0099] A white crystalline solid, m.p. 151-152.degree. C. IR (ATR)
u.sub.max cm.sup.-1: 2941, 1702 (C.dbd.O), 1634 (CONH), 1508, 1409,
1227, 1189, 1025, 880, 820. .sup.1H NMR (600 MHz, CDCl.sub.3):
.delta. 0.93 (3H, s, Me-25), 0.94 (1H, m, H-12), 1.02 (6H, s,
Me-26, 27), 1.03 (3H, s, Me-24), 1.06 (3H, s, Me-23), 1.25-1.48
(13H, m, H-1, 5, 6, 6, 7, 7, 9, 11, 11, 15, 16, 21, 22), 1.19 (1H,
m, H-15), 1.58 (1H, m, H-12), 1.59 (1H, t, J=11.4 Hz, H-18), 1.68
(3H, s, Me-30), 1.74 (1H, m, H-22), 1.89 (2H, m, H-1, 21), 2.16
(1H, m, H-16), 2.39 (1H, m, H-2), 2.49 (1H, m, H-2), 2.98 (6H, m,
H-13, 19, 3', 3', 5', 5'), 3.76 (4H, m, H-2', 2', 6', 6'), 4.74 and
4.57 (2H, both br. s, H-29), 6.89 (2H, m, H-2'', 6''), 6.98 (2H, m,
H-3'', 5'').
Example 3
[0100] 0.68 g (1.44 mmol) of betulonic acid chloride was dissolved
in 35 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.8 mL (5.76 mmol) of anhydrous triethylamine and 0.67 g (2.88
mmol) of 1-(3,4-dichlorophenyl) piperazine were added thereto, and
then stirred at 25.degree. C. for 48 hours. The resulting product
was washed with 10% HCl (40 mL.times.3), further washed with pure
water (40 mL.times.3), and then a solvent layer was dried by 10 g
of anhydrous Na.sub.2SO.sub.4. A dichloromethane (DCM) layer was
evaporated and dried to obtain 0.97 g of light brown solids. These
light brown solids were dissolved in ethanol to prepare a sample,
and then this sample was fractionated by RP C.sub.18 semi-prep
HPLC, and then completely evaporated and dried to obtain 0.83 g
(yield: 86.7%) of 4-(3',4'-dichlorophenylpiperazine-1-yl) amide
betulonic acid.
[0101] C.sub.40H.sub.56Cl.sub.2N.sub.2O.sub.2,
4-(3',4'-dichlorophenylpiperazine-1-yl) amide betulonic acid 3
##STR00007##
[0102] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0103] A white crystalline solid, m.p. 182-183.degree. C. IR (ATR)
u.sub.max cm.sup.-1: 2941, 1700 (C.dbd.O), 1626 (CONH), 1593, 1483,
1411, 1232, 1192, 1026, 882, 801. .sup.1H NMR (600 MHz,
CDCl.sub.2): .delta. 0.93 (3H, s, Me-25), 0.94 (1H, m, H-12), 0.97
(6H, s, Me-26, 27), 1.01 (3H, s, Me-24), 1.06 (3H, s, Me-23), 1.19
(1H, m, H-15), 1.30-1.48 (13H, m, H-1, 5, 6, 6, 7, 7, 9, 11, 11,
15, 16, 21, 22), 1.58 (2H, m, H-12, 18), 1.68 (3H, s, Me-30), 1.74
(1H, m, H-22), 1.89 (2H, m, H-1, 21), 2.14 (1H, m, H-16), 2.39 (1H,
m, H-2), 2.49 (1H, m, H-2), 2.91 (1H, m, H-13), 2.99 (1H, m, H-19),
3.11 (4H, m, H-3', 3', 5', 5'), 3.76 (4H, m, H-2', 2', 6', 6'),
4.74 and 4.59 (2H, both br. s, H-29), 6.76 (1H, dd, J.sub.1=8.4 Hz,
J.sub.2=2.4 Hz, H-6''), 6.96 (1H, d, J=2.4 Hz, H-5''), 7.29 (1H, m,
H-2'').
Example 4
[0104] 0.8 g (1.69 mmol) of betulonic acid chloride was dissolved
in 35 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.94 mL (6.76 mmol) of anhydrous triethylamine and 0.7 g (3.38
mmol) of 1-(4-nitrophenyl)piperazine were added thereto, and then
stirred at 25.degree. C. for 48 hours. The resulting product was
washed with 10% HCl (40 mL.times.3), further washed with pure water
(40 mL.times.3), and then a solvent layer was dried by 10 g of
anhydrous Na.sub.2SO.sub.4. A dichloromethane (DCM) layer was
evaporated and dried to obtain 1.1 g of light brown solids. These
light brown solids were dissolved in ethanol to prepare a sample,
and then this sample was fractionated by RP C.sub.18 semi-prep
HPLC, and then completely evaporated and dried to obtain 0.86 g
(yield: 78.5%) of 4-(4'-nitrophenylpiperazine-1-yl) amide betulonic
acid.
[0105] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0106] A light yellow crystalline solid, m.p. 197-198.degree. C. IR
(ATR) u.sub.max cm.sup.-1: 2940, 1700 (C.dbd.O), 1626 (CONH), 1593,
1501, 1328, 1243, 1190, 1113, 1024, 879, 835. .sup.1H NMR (600 MHz,
CDCl.sub.3): .delta. 0.93 (3H, s, Me-25), 0.96 (1H, m, H-12), 0.98
(3H, s, Me-27), 0.99 (3H, s, Me-26), 1.03 (3H, s, Me-24), 1.08 (3H,
s, Me-23), 1.20 (1H, m, H-15), 1.30-1.52 (14H, m, H-1, 5, 6, 6, 7,
7, 9, 11, 11, 12, 15, 16, 21, 22), 1.61 (1H, t, J=11.4 Hz, H-18),
1.69 (3H, s, Me-30), 1.75 (1H, m, H-22), 1.89 (2H, m, H-1, 21),
2.14 (1H, m, H-16), 2.39 (1H, m, H-2), 2.49 (1H, m, H-2), 2.90 (1H,
m, H-13), 3.00 (1H, dt, J.sub.1=10.8 Hz, J.sub.2=4.2 Hz, H-19),
3.41 (4H, m, H-3', 3', 5', 5'), 3.80 (4H, m, H-2', 2', 6', 6'),
4.74 and 4.60 (2H, both br. s, H-29), 6.85 (2H, d, J=9.6 Hz, H-2'',
6''), 8.14 (2H, d, J=9.0 Hz, H-3'', 5'').
[0107] C.sub.40H.sub.57N.sub.3O.sub.4,
4-(4'-nitrophenylpiperazine-1-yl) amide betulonic acid 4
##STR00008##
Example 5
[0108] 0.77 g (1.63 mmol) of betulonic acid chloride was dissolved
in 40 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.65 mL (6.52 mmol) of anhydrous triethylamine and 0.68 g (3.26
mmol) of 1-(2-pyridyl) piperazine were added thereto, and then
stirred at 25.degree. C. for 48 hours. The resulting product was
washed with 10% HCl (40 mL.times.3), further washed with pure water
(40 mL.times.3), and then a solvent layer was dried by 10 g of
anhydrous Na.sub.2SO.sub.4. A dichloromethane (DCM) layer was
evaporated and dried to obtain 1.0 g of light brown solids. These
light brown solids were dissolved in ethanol to prepare a sample,
and then this sample was fractionated by RP C.sub.18 semi-prep
HPLC, and then completely evaporated and dried to obtain 0.85 g
(yield: 87.6%) of white crystalline 4-(2-pyridyl
piperazine-1-yl)amide betulonic acid.
[0109] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0110] A white crystalline solid, m.p. 158-159.degree. C. IR (ATR)
u.sub.max cm.sup.-1: 2938, 1703 (C.dbd.O), 1633 (CONH), 1592, 1478,
1435, 1243, 1189, 1020, 980, 882, 773. .sup.1H NMR (600 MHz,
CDCl.sub.3): .delta. 0.93 (3H, s, Me-25), 0.95 (1H, m, H-12), 0.98
(6H, s, Me-26, 27), 1.01 (3H, s, Me-24), 1.06 (3H, s, Me-23), 1.19
(1H, m, H-15), 1.30-1.50 (14H, m, H-1, 5, 6, 6, 7, 7, 9, 11, 11,
12, 15, 16, 21, 22), 1.59 (1H, m, H-18), 1.69 (3H, s, Me-30), 1.74
(1H, s, H-22), 1.89 (2H, m, H-1, 21), 2.03 (1H, m, H-16), 2.39 (1H,
m, H-2), 2.49 (1H, m, H-2), 2.94 (1H, m, H-13), 3.00 (1H, td,
J.sub.1=10.8 Hz, J.sub.2=3.6 Hz, H-19), 3.51 (4H, m, H-3', 3', 5',
5'), 3.73 (4H, m, H-2', 2', 6', 6'), 4.74 and 4.59 (2H, both br. s,
H-29), 6.68 (2H, m, H-3'', 5''), 7.52 (1H, m, H-4''), 8.20 (1H, m,
H-6'').
[0111] C.sub.39H.sub.57N.sub.3O.sub.2, 4-(2-pyridyl
piperazine-1-yl) amide betulonic acid 5
##STR00009##
Example 6
[0112] 0.75 g (1.54 mmol) of betulonic acid chloride was dissolved
in 40 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.62 mL (6.16 mmol) of anhydrous triethylamine and 0.52 g (3.08
mmol) of 1-(2-pyrimidyl)piperazine were added thereto, and then
stirred at 25.degree. C. for 48 hours. The resulting product was
washed with 10% HCl (40 mL.times.3), further washed with pure water
(40 mL.times.3), and then a solvent layer was dried by 10 g of
anhydrous Na.sub.2SO.sub.4. A dichloromethane (DCM) layer was
evaporated and dried to obtain 1.05 g of light brown solids. These
light brown solids were dissolved in ethanol to prepare a sample,
and then this sample was fractionated by RP C.sub.18 semi-prep
HPLC, and then completely evaporated and dried to obtain 0.98 g
(yield: 77.8%) of 4-(2-pyrimidyl piperazine-1-yl) amide betulonic
acid.
[0113] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0114] A white crystalline solid, m.p. 109-110.degree. C. IR (ATR)
u.sub.max cm.sup.-1: 2940, 1703 (C.dbd.O), 1633 (CONH), 1584, 1448,
1257, 1188, 982, 879, 797. .sup.1H NMR (600 MHz, CDCl.sub.3):
.delta. 0.93 (3H, s, Me-25), 0.95 (1H, m, H-12), 0.98 (6H, s,
Me-26, 27), 1.02 (3H, s, Me-24), 1.06 (3H, s, Me-23), 1.20 (1H, m,
H-15), 1.30-1.50 (13H, m, H-1, 5, 6, 6, 7, 7, 9, 11, 11, 15, 16,
21, 22), 1.59 (2H, m, H-12, 18), 1.69 (3H, s, Me-30), 1.75 (1H, m,
H-22), 1.89 (2H, m, H-1, 21), 2.16 (1H, m, H-16), 2.39 (1H, m,
H-2), 2.50 (1H, m, H-2), 2.94 (1H, m, H-13), 3.00 (1H, dt,
J.sub.1=10.8 Hz, J.sub.2=3.6 Hz, H-19), 3.69 (4H, m, H-3', 3', 5',
5'), 3.76 (4H, m, H-2', 2', 6', 6'), 4.74 and 4.59 (2H, both br. s,
H-29), 6.55 (1H, m, H-5''), 8.33 (2H, m, H-4'', 6'').
[0115] C.sub.38H.sub.56N.sub.4O.sub.2, 4-(2-pyrimidyl
piperazine-1-yl) amide betulonic acid 6
##STR00010##
Example 7
[0116] 0.61 g (1.29 mmol) of betulonic acid chloride was dissolved
in 40 mL of anhydrous dichloromethane (CH.sub.2Cl.sub.2), and then
0.72 mL (5.16 mmol) of anhydrous triethylamine and 0.60 g (2.58
mmol) of 1-(4-trifluoromethylphenyl)piperazine were added thereto,
and then stirred at 25.degree. C. for 48 hours. The resulting
product was washed with 10% HCl (40 mL.times.3), further washed
with pure water (40 mL.times.3), and then a solvent layer was dried
by 10 g of anhydrous Na.sub.2SO.sub.4. A dichloromethane (DCM)
layer was evaporated and dried to obtain 0.85 g of light brown
solids. These light brown solids were dissolved in ethanol to
prepare a sample, and then this sample was fractionated by RP
C.sub.18 semi-prep HPLC, and then completely evaporated and dried
to obtain 0.64 g (yield: 74.9%) of
4-(4'-trifluoromethylphenylpiperazine-1-yl) amide betulonic
acid.
[0117] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0118] A white crystalline solid, m.p. 229-230.degree. C. IR (ATR)
u.sub.max cm.sup.-1: 2944, 1704 (C.dbd.O), 1616 (CONH), 1525, 1458,
1332, 1230, 1112, 1073, 1023, 885, 830. .sup.1H NMR (600 MHz,
CDCl3): 0.93 (3H, s, Me-25), 0.96 (1H, m, H-12), 0.97 (3H, s,
Me-27), 0.98 (3H, s, Me-26), 1.02 (3H, s, Me-24), 1.06 (3H, s,
Me-23), 1.19 (1H, m, H-15), 1.30-1.52 (12H, m, H-1, 5, 6, 6, 7, 7,
9, 11, 11, 15, 16, 21), 1.59 (2H, m, H-12, 18), 1.69 (3H, s,
Me-30), 1.75 (1H, m, H-22), 1.89 (2H, m, H-1, 21), 2.01 (1H, m,
H-22), 2.16 (1H, m, H-16), 2.39 (1H, m, H-2), 2.49 (1H, m, H-2),
2.92 (1H, m, H-13), 3.00 (1H, dt, J.sub.1=11.4 Hz, J.sub.2=4.2 Hz,
H-19), 3.41 (4H, m, H-3', 3', 5', 5'), 3.78 (4H, m, H-2', 2', 6',
6'), 4.74 and 4.60 (2H, both br. s, H-29), 6.94 (2H, d, J=8.4 Hz,
H-2'', 6''), 7.51 (2H, d, J=8.4 Hz, H-3'', 5'').
[0119] C.sub.41H.sub.59N.sub.2O.sub.2F.sub.3,
4-(4'-trifluoromethylphenylpiperazine-1-yl) amide betulonic acid
7
##STR00011##
[0120] .sup.13C and .sup.1H NMR spectra (respectively, 125 and 600
MHz) of samples dissolved in CDCl.sub.3 were analyzed using a
Varian UNITY INOVA 600 spectrometer, and thus the chemical shifts
of the samples were evaluated by calculating the 5 (ppm) values of
the samples to tetramethylsilane (TMS), and the molecular
structures of the samples were observed by .sup.13C NMR, as given
in Table 2 below.
TABLE-US-00002 TABLE 2 .sup.13C NMR spectral data of BOA, BOCl and
six synthesized compounds (125 MHz, CDCl.sub.3) C atom 1 2 3 4 5 6
7 C-1 39.84 39.89 39.88 39.88 39.88 39.87 39.89 C-2 34.31 34.39
34.38 34.36 34.37 34.37 34.39 C-3 218.32 218.50 218.50 218.42
218.47 218.45 218.54 C-4 47.55 47.57 47.57 47.56 47.55 47.54 47.58
C-5 55.19 55.27 55.26 55.25 55.26 55.26 55.26 C-6 19.80 19.84 19.83
19.83 19.84 19.83 19.83 C-7 33.76 33.88 33.87 33.88 33.88 33.87
33.87 C-8 40.86 40.81 40.81 40.81 40.81 40.80 40.81 C-9 50.13 50.41
50.40 50.38 50.41 50.40 50.40 C-10 37.11 37.15 37.15 37.14 37.14
37.14 37.15 C-11 21.57 21.87 21.86 21.85 21.87 21.87 21.86 C-12
25.54 25.85 25.83 25.82 25.86 25.85 25.83 C-13 37.98 37.19 37.19
37.21 37.19 37.19 37.19 C-14 42.69 42.16 42.16 42.18 42.16 42.16
42.17 C-15 29.76 30.01 30.01 30.03 30.01 30.01 30.01 C-16 32.37
33.88 33.87 33.88 33.88 33.87 33.87 C-17 67.96 54.76 54.76 54.80
54.80 54.82 54.77 C-18 49.79 52.78 52.75 52.73 52.80 52.81 52.74
C-19 46.16 45.83 45.82 45.83 45.85 45.84 45.84 C-20 149.43 151.41
151.32 151.19 151.42 151.41 151.34 C-21 30.02 31.50 31.48 31.46
31.50 31.50 31.47 C-22 36.37 36.19 36.18 36.17 36.21 36.22 36.20
C-23 26.78 26.79 26.79 26.79 26.79 26.78 26.78 C-24 21.23 21.24
21.24 21.22 21.22 21.22 21.24 C-25 16.18 16.23 16.22 16.21 16.21
16.24 16.23 C-26 15.92 16.13 16.12 16.12 16.12 16.20 16.13 C-27
14.91 14.82 14.82 14.82 14.81 14.81 14.83 C-28 177.59 173.73 173.79
173.99 173.85 173.90 173.83 C-29 110.552 109.50 109.56 109.63
109.47 109.48 109.57 C-30 19.54 19.88 19.85 19.82 19.86 19.87 19.86
C-2' 32.75 32.74 32.71 32.72 32.74 32.74 C-3' 50.81 49.29 47.37
45.82 44.15 48.56 C-5' 50.81 49.29 47.37 45.82 44.15 48.56 C-6'
32.75 32.74 32.71 32.72 32.74 32.74 C-1'' 147.84 150.59 154.86
153.25 C-2'' 118.38 130.75 113.19 159.57 161.956 115.06 d, (6.38
Hz).sup.a C-3'' 115.87 133.10 126.11 107.70 126.67 d, (18.37
Hz).sup.a q (3.25 Hz).sup.a C-4'' 157.7 123.13 139.25 137.90 157.94
121.43 d, (199.1 Hz).sup.a q (27.0 Hz).sup.a C-5'' 115.87 117.81
126.11 114.21 110.68 126.67 d, (18.37 Hz).sup.a q (3.25 Hz).sup.a
C-6'' 118.38 115.91 113.19 148.06 157.94 115.06 d, (6.38 Hz).sup.a
C-7'' 124.80 q (224.38 Hz).sup.a .sup.aValues in parentheses are
coupling constants in Hz with fluorine.
Example 8
[0121] An antibacterial test was carried out using the
above-mentioned paper disc diffusion method. Betulonic acid amide
was dissolved in DMSO to make a sample having a concentration of 1
mg/mL. A filter paper (diameter: 8 mm, manufactured by Advantec,
Toyo Roshi Kaisha, Japan) wetted with 60 .mu.L of the sample was
attached to an agar plate, and then a circumference, around which
bacteria were not cultivated, was measured. DMSO was used as a
negative control, and gentamicin was used as a positive control.
The antibacterial test was repeatedly carried out three times.
Ochrobactrum anthropi KCTC 22833T and Stenotrophomonas maltophilia
KCTC 1773T used in the antibacterial test were purchased from
Korean Collection for Type Culture (KCTC), and Ochrobactrum
intermedium KACC 11952T and Stenotrophomonas nitritireducens KACC
10891T used in the antibacterial test were purchased from Korean
Agricultural Culture Collection (KACC). The diameters of clear
circular zones of samples 2 to 7 of FIG. 1 were measured by a paper
disc diffusion method, and compared to that of gentamicin. The
results thereof are given in Table 3 below. From Table 3, it can be
ascertained that sample 1 (4-(4'-fluorophenylpiperazin-1-yl) amide
betulonic acid) provides a growth inhibition effect only to
Stenotrophomonas nitritireducens, but other samples 2 to 7 provides
growth inhibition effects to four kinds of bacteria. Particularly,
it can be ascertained that the activity of sample 2
(4-(3',4'-dichlorophenylpiperazin-1-yl) amide betulonic acid) and
the activity of sample 7
(4-(4'-trifluoromethylphenylpiperazin-1-yl) amide betulonic acid)
are highest.
TABLE-US-00003 TABLE 3 Results of antibacterial test of betulonic
acid amides Antibacterial zone of inhibition (mm) O. O. S. S.
Compound anthropi intermedium nitritireducens maltophila 1 of FIG.
1 -- -- 11 -- 2 of FIG. 1 15 14 15 13 3 of FIG. 1 12 13 13 11 4 of
FIG. 1 12 10 11 10 5 of FIG. 1 13 10 11 10 6 of FIG. 1 15 14 16 14
Gentamicin 19 17 21 16
Example 9
[0122] Step (I): Synthesis of
N.alpha.-butyloxycarbonyl-N.epsilon.-benzyloxycarbonyl-Lysin methyl
ester (Boc-Lys(Cbz)-OMe) referred to the paper [Kobayashi et al.,
J. Org. Chem. 66:6626-6623(2001)]. 2 g of
N.alpha.-butyloxycarbonyl-N.epsilon.-benzyloxycarbonyl-Lysin
(Boc-Lys(Cbz)-OH) was dissolved in 15 mL of
trimethylsilydiazomethane, 10 mL of anhydrous methanol was added
thereto, and then stirred at room temperature for 60 min. The
reaction product was passed through 7.7 g of silica gel (Sigma
Aldrich, Silical Davisil Grade 635, pore 60, 60-100 mesh) to obtain
a filtrate. The filtrate was evaporated to obtain a light yellow
N.alpha.-butyloxycarbonyl-N.epsilon.-benzyloxycarbonyl-Lysin methyl
ester (Boc-Lys(Cbz)-OMe).
[0123] Step (II): A total amount of Boc-Lys(Cbz)-OMe was dissolved
in a mixed solvent of 40 ml of ethyl acetate and 40 ml of methanol,
and then the resultant solution was stirred at room temperature for
4 hours under a hydrogen atmosphere while gradually adding 0.223 g
of Pd/C. The reaction product was passed through 3.56 g of celite
(Sigma Aldrich) to remove Pd/C, and was then washed with 20 ml of
methanol (MeOH) two times to obtain a filtrate. The filtrate was
evaporated to obtain light viscous solid Boc-Lys-OMe.
[0124] Step (III): The synthesis reaction of betulonic acid and
Boc-Lys-OMe referred to the paper [Zhao et al., J. Org. Chem.
69:270-279(2004)]. A total amount of the Boc-Lys-OMe obtained in
step (II) and 2 g of betulonic acid were dissolved in 60 ml of THF
(tetrahydrofuran), and then 1127 mg of
1,3-dicyclohexylecarbodiimide (DCC), 754 mg of
1-hydroxybenzotriazole hydrate (HOBt) and 0.925 ml of triethylamine
were added thereto. The resultant solution was stirred for 2 hours
while maintaining the temperature of a reactor at 0.degree. C.
[0125] When a white precipitate was generated in the reactor, the
temperature of the reactor was maintained at room temperature, and
the synthesis reaction of a Boc-Lys-BOA monomer ester was
conducted. 0.2 ml of the synthesis reaction product was completely
evaporated, dried and then dissolved in 0.94 ml of ethanol (EtOHD)
to obtain a sample. The concentration of Boc-Lys-BOA monomer ester
was analyzed while analyzing the sample using HPLC.
[0126] The sample extracted from the reactor was analyzed for 62
hours using a HPLC gradient elution method of Table 1 until the
peak area of a component (retention time: 12.0 min) corresponding
to Boc-lysinated betulonic acid monomer ester was not changed.
[0127] As the result of analysis of the sample, the sample includes
impurities having an area ratio of the sample to Boc-Lys-BOA
monomer ester of 7.85 and retention times of 3.5, 4.1, 15.15, 16.0
and 23.23 min in amounts of 50.39%, 3.1%, 0.68%, 32.68%, 2.97% and
2.31%, respectively (refer to FIG. 3).
Example 10
[0128] Purification of organic synthesis reaction product of
Boc-Lysinated betulonic acid monomer ester and purification of
hydrolysis reaction product of Boc-Lysinated betulonic acid monomer
ester
[0129] 3.37 g of white crystal (55), which is an organic synthesis
reaction product of Boc-Lysinated betulonic acid mono ester, was
dissolved in 600 ml of a mixed solvent of EtOAc:hexane (1:1, v/v)
to make a sample (56), and then the sample (56) was passed through
a column (90) filled with 40 g of aluminum oxide to decrease the
concentration of a polar component having a retention time of 4 min
to 5 min from 50.39% to 4.86.about.6.96%. 80 ml of a mixed solvent
(57) of EtOAc:hexane (1:1, v/v) was supplied to an adsorption tower
(90) to wash the sample. The solution (58) discharged from the
adsorption tower (90) and the solution (59) discharged from a
washing tower were evaporated and dried to obtain 2.825 g of white
crystals (60), and then the white crystals (60) were treated with
aluminum oxide to increase the concentration of Boc-Lysinated
betulonic acid monomer ester from 7.85% to 14.6%. 2.825 g of the
white crystals (60) were dissolved in 200 ml of ethanol (EtOH) to
make a sample (61), and then the sample (61) was filtered to
separate an insoluble white precipitate and remove 0.8096 g of a
component (62) having a retention time of 15 min to obtain a first
filtrate (63), and then ethanol (EtOH) was evaporated until the
volume of the first filtrate (63) became 75 ml. Then, the first
filtrate (63) was further filtered to remove 0.1134 g of a
component (65) having a retention time of 15 min to obtain a second
filtrate (66), and then ethanol (EtOH) was evaporated until the
volume of the second filtrate (66) became 25 ml. Then, the second
filtrate (66) was further filtered to remove 0.1324 g of a
component (68) having a retention time of 15 min to obtain a third
filtrate (69), and then the third filtrate (69) was completely
evaporated to obtain 1.1845 g of white crystals (71), thus
increasing the concentration of Boc-Lysinated betulonic acid
monomer ester from 14.6% to 34.33%.
[0130] The white crystals (71) were dissolved in 25 ml of ethanol
to make a sample (72), and then the sample (72) was introduced into
a RP C.sub.18 semi-preparative chromatography column (eluent
MeCN:H.sub.2O=86:14, v/v, 7 ml/min, 250 mm.times.21.1 mm) to
collect a Boc-Lysinated betulonic acid monomer ester fraction, and
then this Boc-Lysinated betulonic acid monomer ester fraction was
evaporated and dried to obtain 0.6597 g of white powder (75)
(referring to FIG. 4, Boc-Lys-BOA monomer ester purity >99%,
yield to birch bark: 0.58 wt %).
[0131] 256.1 mg of the white powder (75) having a Boc-Lys-BOA
monomer ester purity of 99% or more was dissolved in 5 ml of
tetrahydrofuran (THF), 18.28 mg of LiOH monohydrate and 0.767 ml of
pure water were added thereto, and then the resultant solution was
stirred at room temperature for 7 hours to hydrolyze methyl ester
bonded to C-terminus of lysine. The hydrolysis reaction product
(76) was evaporated and dried to obtain white solids (77). The
obtained white solids (77) was dissolved in 15 ml of ethanol (EtOH)
having the lowest LiOH solubility to make a sample (78). The sample
(78) was filtered by a PTFE membrane and washed with 5 ml of
-20.degree. C. ethanol (EtOH) to remove insoluble LiOH (79) to
obtain a filtrate (80). 20 ml of the filtrate (80) was introduced
into a RP C.sub.18 semi-preparative chromatography column (250
mm.times.21.1 mm, 10 .mu.m, 100 .ANG., MeCN:H.sub.2O=86:14 v/v, 7
ml/min). The solution discharged from the column was detected by a
UV/VIS detector at 212 nm and 252 nm, and simultaneously a
Boc-Lysinated betulonic acid fraction was separated from the
solution. The Boc-Lysinated betulonic acid fraction was evaporated
and dried to obtain white crystals (referring to FIG. 4,
Boc-Lys-BOA monomer ester purity >99.5%, yield to birch bark:
0.47 wt %).
[0132] C.sub.42H.sub.68N.sub.2O.sub.6: Boc-lysinated betulonic acid
monomer ester
[0133] .sup.1H NMR (600 MHz, CDCl.sub.3):
[0134] 0.93 (3H, s, Me-25), 0.96 (3H, s, Me-27), 0.97 (3H, s,
Me-26), 1.01 (1H, M, H-12), 1.02 (3H, s, Me-24), 1.06 (3H, s,
Me-23), 1.06 (3H, s, Me-23), 1.14-1.58 (18H, m, H-1, 5, 6, 6, 7, 7,
9, 11, 11, 12, 15, 15, 16, 18, 21, 22, 33, 33), 1.45 (9H, br. s,
H-40), 1.68 (3H, s, H-30), 1.73 (4H, m, H-1, 16, 21, 22), 1.91 (4H,
m, H-32, 34), 2.39 (1H, m, H-2), 2.49 (2H, m, H-2, 13), 3.14 (1H,
dt, J=11.4 Hz, J=4.8 Hz, H-19), 3.20 (1H, m, H-31), 3.25 (1H, m,
H-31), 3.74 (3H, s, H-37), 4.29 (1H, m, H-35), 4.29 (1H, m, H-35),
4.74 and 4.59 (2H, both br. s, H-29), 5.06 (1H, d, J=7.8 Hz, H-42),
5.69 (1H, t, J=5.4 Hz, H-41).
TABLE-US-00004 TABLE 4 .sup.13C NMR of Boc-Lysinated betulonic acid
monomer ester C BOA(betulonic acid) Boc-Lys-BOA monomer ester C-1
39.80 39.04 C-2 34.33 34.37 C-3 218.55 218.54 C-4 47.54 47.55 C-5
55.62 55.18 C-6 19.82 19.83 C-7 33.78 33.88 C-8 40.82 39.83 C-9
50.03 50.18 C-10 37.25 37.10 C-11 21.56 22.92 C-12 26.67 25.79 C-13
38.71 37.93 C-14 42.68 42.70 C-15 30.75 29.58 C-16 32.31 33.95 C-17
56.62 55.72 C-18 49.36 50.23 C-19 47.10 46.88 C-20 150.52 151.11
C-21 29.88 31.02 C-22 37.11 38.63 C-23 26.84 26.77 C-24 21.20 21.23
C-25 16.17a 16.19 C-26 16.02a 16.16 C-27 14.83 14.75 C-28 182.80
177.97 C-29 109.98 109.60 C-30 19.57 19.68 C-31 40.86 C-32 31.02
C-33 21.64 C-34 32.68 C-35 53.43 C-36 173.47 C-37 52.54 C-38 155.63
C-39 80.12 C-40 28.52
[0135] Although the preferred embodiments of the present invention
have been disclosed for illustrative purposes, those skilled in the
art will appreciate that various modifications, additions and
substitutions are possible, without departing from the scope and
spirit of the invention as disclosed in the accompanying
claims.
* * * * *